Chromatin structure provides a necessary compaction to the eukaryotic genome, yet it also impairs protein access to DNA, thus imposing additional regulation on protein-DNA transactions. The overall objective of this project is to understand how chromatin controls gene expression. This application focuses on an essential and conserved transcription elongation factor, Spt6, which has critical and widespread roles in transcription elongation: Mutations in Spt6 has been shown to cause developmental defects in higher eukaryotes including C. elegans and zebrafish. In addition, Spt6 has been shown to regulate transcription of HIV-1 viral genes and genes of the human cytomegalovirus which infects immunosuppressed patients and newborns. Taken together, these findings highlight the importance of Spt6 in regulating chromatin structure and transcription during development and in human diseases, yet the molecular mechanism by which Spt6 functions is largely unknown. This study proposes biochemical and genomic approaches to understand how Spt6 regulates transcription. The TAP-tag purification of Spt6 in Aim 1 will comprehensively identify Spt6-interacting proteins in vivo. The focus will be on the interaction between Spt6 and histones and how the histone code regulates transcription elongation. The biochemical experiments proposed in Aim 2 will elucidate how Spt6 interacts with nucleosomes to alter chromosomal structure during transcription elongation and restore proper structure after transcription. The main focus will be on the role of Spt6 in transcription-dependent histone exchange. Finally the genome-wide ChlP-chip study proposed in Aim 3 will identify the binding locations of Spt6, and correlate them with existing genomic data such as transcription activity in known ORFs, spt6 expression profile, and histone modifications maps. The goal is to define all the cellular processes regulated by Spt6, and focus on novel targets of Spt6. The proposed studies will provide further insights into the role of Spt6 in DNA metabolic activities regulated by chromatin structure. Relevance: Disturbances in the process of transferring information from genes to proteins, known as transcription, are involved in many human diseases such as cancer, heart disease, and inflammation. The goal of this research is to elucidate the function of Spt6, an essential and evolutionarily conserved protein that has critical and widespread roles in transcription.